Method for screening potential therapeutically effective antiviral agents

ABSTRACT

The invention provides methods for screening agents for potential anti-viral effects by assessing the ability of the agents to suppress viral replication and/or pathology in thymic cells grown in thymic organ culture in vitro. Also provided are methods to study viral pathology and infectivity.

This application is a division of application Ser. No. 08/109,305 filedAug. 19, 1993.

DESCRIPTION

1. Technical Field

The present invention is directed to methods to screen potentialtherapeutic anti-viral agents, particularly those that suppress viralreplication and/or pathology in thymus cells.

2. Background Art

The ability to obtain therapeutically effective anti-viral agents isfacilitated by preclinical screening systems that are relevant,predictive and amenable to high throughput. Such systems include invitro cell-free assays, in vitro tissue culture assays and assays inanimal models. Important animal models have traditionally includedlaboratory mice, rats and non-human primates. More recently,specifically modified mice such as SCID-hu mice and hu-PBL mice havealso played a role. However, even these higher-order models do notusually reflect human physiology in its entirety. Another drawback ofanimal testing is the need for extensive and direct contact withinfected animals and, therefore, the potential exposure to drugs andviruses. This situation is particularly undesirable in the case of humanimmunodeficiency virus (HIV). Furthermore, animal systems are usuallytime consuming and expensive, making them unsuitable for rapid screeningof large numbers of potential therapeutic agents.

For numerous viruses, there are no animal models; thus efficacy testingof putative therapeutic agents on human beings occurs only at theclinical stage. The extreme expense and safety concerns of this clinicaltesting pose a barrier to the current development of therapeutics andultimate progress in delineating the mechanisms of viral infectivity andpathogenesis.

Although in vitro drug screening systems exist, drug efficacy resultsfrom in vitro cell-free and cell culture systems do not always correlatewell with in vivo efficacy. Nevertheless, in vitro systems are essentialin modern pharmaceutics for large-scale screening of agents withtherapeutic potential. The more closely an in vitro system mimics invivo infection, the more useful the in vitro assay. Thus, in vitro drugscreening assays are constantly being developed to more closely mimic invivo responses.

HIV is the name given to a group of highly related viruses which hasbeen identified as the primary etiological agent of the acquiredimmunodeficiency syndrome (AIDS) and AIDS-related complex (ARC) inhumans. HIV is a deadly infectious disease and a major worldwide healthproblem. Progress towards understanding the pathogenesis of HIVinfection in vivo and discovering therapeutic agents has been hamperedby a lack of a cost-effective, efficient, and clinically relevant invitro screening system. One of the most widely-used animal models forHIV infection is the rhesus macaque and infection with simianimmunodeficiency virus (SIV). In this system, large amounts of drugsmust be synthesized and isolated prior to screening, an expensive andtime consuming task. Maintaining the animals is expensive. Because ofthe high cost of the monkey model, it is only used to test drugs alreadyshowing strong indications of success in in vitro testing. Moreover, themonkey model utilizes a different, although related, virus, and it isnot necessarily true that antiviral compounds active against SIV will beactive against HIV, and vice versa.

What is needed to develop anti-viral agents is an effective in vitroscreening system that reflects in vivo pathogenesis and which is capableof rapidly screening a wide variety of agents. Such an in vitro systemshould provide the reliability of the more expensive in vivo systems andyet afford rapid, cost-effective initial screening for potentialtherapeutics from a virtually limitless variety of sources. To theextent that such a system is relevant to HIV infection in vivo, itshould also be useful for the evaluation of pathogenic mechanismsassociated with HIV infection.

DISCLOSURE OF INVENTION

The present invention is directed to a method of screening agents fortherapeutic potential. The method comprises incubating thymic lobules,preferably human fetal thymic lobules, for a time and under conditionssufficient to allow viral infection. The lobules are then exposed to avirus, and, either prior to virus exposure, simultaneously, or shortlythereafter, exposed to an agent to be screened. The lobule contents arethen assayed for suppression of viral infection. Therapeutic potentialis present if any indicia of viral infection are diminished relative toa control sample that has not been exposed to the agent.

The method can also be used to study viral transmission, infection,replication, and pathogenesis.

DEFINITIONS

The following definitions refer to terms and phrases used herein. Otherdefinitions appear as indicated.

Suppression indicates that there is evidence of reduced viraltransmission, infection, replication, or pathology.

Assaying cells for viral activity is done by observing any factors suchas cell viability and yields and CD4 and CD8 phenotypes.

A control sample is one in which thymic lobules are obtained andincubated under identical conditions as the lobules to be infected butnot exposed to the virus to be tested.

Pathology is viral infection, replication, cell death, slowing oraberrant cell growth or atypical cellular phenotype or function due toviral infection.

An anti-viral agent is one that prevents or treats viral infection,i.e., one that can be used prophylactically or therapeutically. Such ananti-viral agent can affect any of the mechanisms of virus transmission,infection, replication, or pathology.

A potential anti-viral agent is an agent which has been shown tosuppress viral infection and/or pathology in the method describedherein. Such a potential anti-viral agent has an enhanced likelihood ofbeing effective in human prophylaxis and therapy. Potential anti-viralagents can come from a virtually limitless variety of sources.

Viral activity refers to viral infection, re-infection, replication,expression of viral proteins or pathology.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph depicting viable cell yields per incubated thymusfragment in uninfected cultures. FIG. 1 is discussed in Example 1.

FIGS. 2(a) and (b) contain two graphs depicting (a) relative and (b)absolute numbers of CD4+/8+ cells in uninfected cultures. FIG. 2 isdiscussed in Example 1.

FIG. 3 is a graph depicting cell viability of cultures infected with HIVby the standard drip or dip method. FIG. 3 is discussed in Example 3.

FIG. 4 is a graph depicting p24 levels in cultures infected with HIV bythe standard drip or dip method. FIG. 4 is discussed in Example 3.

FIGS. 5(a) and (b) contain two graphs depicting the relative (a) andabsolute (b) numbers of CD4+/8+ cells in cultures infected with HIV bythe standard drip or dip method. FIG. 5 is discussed in Example 3.

FIG. 6 is a graph depicting the number of cells in cultures infected byHIV the standard drip or dip method. FIG. 6 is discussed in Example 3.

FIG. 7 is a graph depicting the relative number of CD8+ cells incultures that have been infected with HIV by the standard drip or dipmethod. FIG. 7 is discussed in Example 3.

FIG. 8 is a graph depicting p24 levels of HIV-infected cultures overtime. FIG. 8 is discussed in Example 4.

FIGS. 9(a) and (b) contain graphs depicting (a) increased cell death and(b) decreased cellularity per thymus fragment in HIV-infected cultures.FIG. 9 is discussed in Example 4.

FIGS. 10(a), (b), (c) and (d) contain a series of graphs depicting in(a) and (c) relative and in (b) and (d) absolute numbers numbers ofviable CD4+/8+ cells in HIV infected cultures. FIG. 10 is discussed inExample 4.

FIG. 11 is a series of graphs depicting fluorescence activated cellsorter (FACS) analysis of HIV-infected cultures. FIG. 11 is discussed inExample 4.

FIG. 12 is a graph depicting p24 levels in HIV-infected cultures treatedwith azidothymidine (AZT) or dideoxyinosine (ddI). FIG. 12 is discussedin Example 6.

FIGS. 13(a) and (b) contain two graphs depicting, (a) relative and (b)absolute numbers of viable CD4+/8+ cells in HIV-infected culturestreated with AZT or ddi. FIG. 13 is discussed in Example 6.

FIG. 14 is a graph depicting relative numbers of CD8+ cells inHIV-infected cultures treated with AZT or ddi. FIG. 14 is discussed inExample 6.

FIG. 15 is a graph depicting p24 levels in HIV-infected cultures (EWstrain) treated with AZT or ddI. FIG. 15 is discussed in Example 6.

FIG. 16 is a graph depicting p24 levels in HIV-infected cultures (NL4-3strain) treated with AZT or ddI. FIG. 16 is discussed in Example 6.

MODES FOR CARRYING OUT THE INVENTION

The present invention is directed to methods of screening agents forpotential anti-viral efficacy and elucidating the mechanisms of viralinfection, transmission, and pathogenesis.

It has now been found that HIV infection of cultures of intact humanthymic lobules mimics the behavior of existing animal models for HIVinfection, including the SCID-hu mouse. The system is also known as TOCfor thymic organ culture. The TOC system is useful for rapid screeningof compounds for anti-viral potential, particularly those for thetreatment and prevention of AIDS. The TOC system is much simpler, safer,faster, and more cost effective than existing animal models. The TOCsystem is also more predictive of the actual efficacy of known anti-HIVcompounds in humans than other in vitro systems.

Utilizing the TOC method described herein, known therapeutic agents havebeen found to exert therapeutic efficacy similar to that found in vivo.More specifically, both 2', 3'-dideoxyinosine (ddI) and azidothymidine(AZT; also referred to as zidovudine) suppressed HIV replication andpathology at equivalent dose ranges found to be active in humans. Infact, the thymic lobule culture system parallels the SCID-hu in vivosystem, but demonstrates faster kinetics. Thus, the results from initialscreening of compounds provided by this invention are more indicative ofactual results in human clinical trials than results frompreviously-described in vitro systems.

In one embodiment, a method is provided to screen agents for potentialanti-viral efficacy. The method comprises providing thymic lobules;incubating the lobules for a time and under conditions sufficient tomaintain cell viability and to allow viral infection; exposing thelobules to a virus under conditions to induce infection; exposing thelobules to an anti-viral agent to be screened; and assaying for evidenceof viral activity.

The agent is a potential anti-viral agent if it suppresses viralpathology relative to lobules incubated in a control sample.

The lobules obtained are thymic. Preferably, the thymic lobules arehuman, including but not limited to neonate and mature human. Morepreferably, the thymic lobules are from human fetal thymus tissues. Thelobule is a relatively intact lymphoid organ subunit that contains manycell types such as thymocytes, macrophages, dendritic cells, andepithelial cells. Thus, it provides a more complex and in vivo-likeenvironment than simpler cell cultures.

Thymic lobules are obtained by dissecting the whole thymus into thymicfragments. Each fragment contains at least one intact thymic lobule. Thefragment is incubated separately under conditions that support cellviability and allow infection of the lobules by the virus. Suitablemethods of cell culture known in the art can be used. In practice, afragment is placed on top of a sterile filter laying across anabsorbable gelatin sponge boat in a tissue culture plate containingsupport media.

Standard cell culture techniques are adequate for use in the methodsdescribed herein. Culture plates are maintained in incubation chambersat 37° C. in an atmosphere of 5% CO₂. Incubation periods prior toinfection may range from essentially no incubation prior to infection toperiods as long as the lobules can be maintained in a viable state andstill retain the ability to be infected by the target virus. Typically,incubation periods range from approximately less than one day to aboutfourteen days. Although the optimal medium varies depending on thelobule source, the type of virus, and the length of the incubationperiod, the preferred medium is one which supports cell viability andallows infection of the lobules by the target virus. Preferably, themedium is Roswell Park Memorial Institute medium (RPMI-1640)supplemented with 10% fetal calf serum (FCS), glutamine, penicillin andstreptomycin.

Successful maintenance of cell viability is dependent on maintenance ofintact lobules and an air interface for cultured lobules. Preferably,medium is changed daily. More preferably, 1X I/S/T(insulin/selenium/transferrin, Sigma) is added as a supplement to themedium to enhance cell viability.

The methods described herein are suitable for use in screening potentialtherapeutic agents against a wide variety of viruses. Candidate viruseswould include those which infect cells found in the thymus. Such virusesinclude but are not limited to human cytomegalovirus, humanimmunodeficiency virus (HIV), herpes simplex virus (HSV), and rubellavirus. The cells found in the thymus include but are not limited tothymocytes, dendritic cells, macrophages, endothelial cells,fibroblasts, B cells, and epithelial cells. Preferably, the viruses arethose which infect thymocytes or cells in the thymic stroma which areimportant for T cell development. More preferably, the viruses arevarious isolates of HIV. Suitable isolates of HIV are those which aremost similar to those which infect human patients. Such isolates includebut are not limited to primary isolates such as EW and infectiousmolecular clones such as JR-CSF, Xho, and NL4-3.

The types of anti-viral agents suitable for testing in this assay arevirtually unlimited. Agents can be obtained from chemical, nutritionaland biological sources. For instance, suitable chemical agents can benovel, untested classes of chemicals, as well as agonists, antagonists,or modifications of known therapeutic agents. Nutritional agents can besimple or complex, ranging from minerals to extracts from plant andanimal sources. Such agents can be easily derived from plant sourcesincluding but not limited to soy, pea, and potato by-products, and fromanimal products such as whey or other milk byproducts. Biological agentsinclude but are not limited to biological response modifiers (such ascytokines), antibodies, and other so-called "small molecules." Suchagents can either be derived from biological sources or chemicallysynthesized to mimic the effect of biological response modifiers.Anti-viral agents may also include those mediating an effect through acellular vehicle. For example, infected, uninfected, transduced, ornon-transduced cells of various phenotypes (such as monocytes,macrophages, epithelial cells) may be introduced, such as bymicroinjection, into the lobules to assess their effect on viralreplication or pathology.

The assay allows rapid screening of potential anti-viral agents, thusenabling the screening of large numbers of different agents at multipleconcentrations. The therapeutic agents tested to date have beeneffective in this assay at concentrations in the sub-micromolar range,indicating that the assay is extremely sensitive. This is a real benefitover current animal systems which require large amounts of agents forscreening. Preferably, the range of concentrations of agents used inthis assay is about 0.001 μM to 5 mM. The upper range is limited by thesolubility of the agent and toxicity of high concentrations of somedrugs; the lower range is not necessarily limited but should be lowenough to encompass a complete dose-response curve.

The lobules can be infected by any method known in the art, includingbut not limited to incubating the lobules in a liquid containing thevirus particles for a length of time suitable to allow infection. Theamount of virus used for infection (reported as infectious dose) dependson the type of virus, the method of infection, the incubation conditionsof the cells, etc. The infectious dose necessary to follow the methodsherein is determined empirically. Such determination is well within theskill of one in the art. A suitable infectious dose is one which allowsmeasurement of a parameter of viral activity and/or infectivity.

In the case of HIV, virus can be added to the lobules by drippingvirus-containing liquid over the top of thymic lobules being incubatedon absorbable gelatin sponge boats. Preferably, however, the thymiclobules are incubated for approximately two hours in vials containingundiluted virus. This is the so-called "dip" method. In the examplesdescribed below, HIV is obtained from phytohemagglutinin(PHA)-stimulated blast cells previously infected by HIV. The "undiluted"virus is simply the supernatant derived from those cells. Infection bydipping the cells in undiluted virus appears to accelerate infection andthe onset of observable HIV pathology.

The anti-viral agents to be assayed are introduced into the lobulesprior to, simultaneously with, or shortly after viral infection. Thetime of introduction will vary according to the type of virus, theincubation conditions and the method of infection. It may be useful totest the same agent at a variety of time points to insure accuratedetermination of efficacy. The optimal conditions in the case of HIV areto pretreat the lobules with the agent approximately 1-2 hours beforeinfection and to incubate the pre-treated, infected lobules in presenceof the agent. After addition of the agent, the lobules are furtherincubated for a time sufficient to allow an assayable parameter of virusinfection or pathology to occur in the control cells.

Tissue fragments are harvested and disrupted to allow the release ofthymocytes and other lobule contents. Extent of viral infection isdetermined by assaying any suitable parameter indicative of viralreplication or viral-induced pathology. These parameters include but arenot limited to: viral-specific DNA, RNA, or protein production,viral-induced cytokine production, thymocyte phenotype profile asdetermined by FACS analysis, histological analyses, apoptotic profile,DNA quantitation, DNA fragmentation profiles, immunohistochemistry,functional assays, PCR analysis of integrated proviral DNA and 2-LTRcircular viral DNA, and isolation of virus in vitro.

A positive response to the anti-viral agents is manifest when cells fromthe lobules treated with the agent exhibit a suppressed level of viralpathology and/or replication compared to infected, untreated controls.The extent of viral replication or pathology is determined by measuringone or more of the various parameters above.

In another embodiment, the present invention is suitable for testinghypotheses concerning mechanisms of viral infection, replication, andpathology in thymic cells. Incubated thymic lobules are infected withthe virus as discussed above. Lobules can be harvested at various timesafter infection, and a variety of viral infection parameters can beobserved. Alternatively, the virus used for infection can bespecifically altered genetically, and the viral activity and replicationcompared to infection with native, non-altered control viruses. Use ofdrugs or other agents with known mechanistic targets can delineate newelements of viral pathology, thereby providing new targets forintervention through cellular therapy, gene therapy, drug therapy, orrational drug design.

The following examples are provided to illustrate but not limit theclaimed invention.

EXAMPLES Example 1 Cell Culture Techniques

Fresh human fetal thymus was obtained at 18-24 weeks gestational age andkept in sterile media at 4° C. Human fetal tissues can be obtained withinformed consent from agencies such as International Institute for theAdvancement of Medicine (Exton, Pa.) or Advance Bioscience Resource(Alameda, Calif.), in compliance with regulations issued by State andFederal governments. Fragments of approximately 1 mm³ were dissectedfrom the fresh thymus. These fragments represent approximately 1-4intact thymic lobules as judged by light microscopy. Fragments aresuitable for this test if they are big enough to contain intact lobulesand small enough to allow diffusion of virus and agents. Typically thisis about 1 mm³.

Fragments were placed on sterile CoStar Nucleopore® filters (13 mm/0.8μm) that lie across Gelfoam® sterile sponge boats (Upjohn) saturated ingrowth media. Growth media was RPMI (1640) with 10% FCS, glutamine,penicillin and streptomycin. Culture plates, typically Cell Wells™,6-well flat bottom 35 mm polystyrene plates (Corning), were maintainedin incubation chambers at 37° C. under 5% CO₂. Preferably, the culturemedium was enriched every 48 hours by removing old medium and thendripping fresh medium over fragments. Incubation periods ranged fromless than one day up to approximately 2 weeks. Typically, a majority ofthe cells of the lobules were viable for approximately 10 days underthese culture conditions.

The number of viable thymocytes from the lobules over a two weekincubation period is shown in FIG. 1. The results show that asignificant loss of thymocytes occurred during culture.

Relative and absolute CD4+/8+ profiles of the cells from the thymiclobule culture are shown in FIGS. 2(a) and 2(b), respectively. Thesefigures showed a depletion in this cell population over the two-weekincubation period, indicating a significant baseline of cell loss/deathin this culture system. Since viral infection can also cause cellloss/death, it was desirable to develop culture conditions thatmaximized thymus fragment cellularity and cell viability.

To optimize cell culture conditions, various media modifications weretested for their ability to enhance overall thymus fragment stability.The culture conditions were modified as follows:

a) variation in FCS concentrations (2-20%);

b) addition of the reducing agent 2-mercaptoethanol (2-ME to a finalconcentration of 50 μM);

c) addition of epidermal growth factor (EGF at 10 ng/ml);

d) addition of 1X supplement containing trace amounts of I/S/T (Sigma).

In the case of 2-ME, EGF, or I/S/T containing media, 10% FCS was used.Uninfected cultures were assayed on day 11 for cell yields, percentviability, and. CD4+/8+ distribution patterns. The results obtained areshown in Table 1. Data represent mean values averaged from threefragments cultured under identical conditions for 11 days.

These data suggest that supplementing media with 1X I/S/T helpedmaintain thymocyte longevity in culture, as evidenced by viability,increased representation of CD4+/8+ thymocytes and increased cell yieldsfollowing 11 days culture. Varying other culture conditions did notproduce any obvious growth or maintenance advantages.

                                      TABLE 1                                     __________________________________________________________________________    Effect of cell culture conditions                                             on thymic cell viability                                                             Cells per                                                                            %   %   %    %   Absolute                                       Culture                                                                              Fragment                                                                             Live                                                                              CD4+                                                                              CD4- CD4+                                                                              Number                                         Condition                                                                            (×10.sup.3)                                                                    Cells                                                                             8-  8+   8+  CD4+8+                                         __________________________________________________________________________    2% FCS 113    27  39  13   39  43000                                          10% FCS                                                                              122    32  31  14   46  59000                                          20% FCS                                                                               83    25  38  12   40  34000                                          2-ME    99    27  47  11   32  34000                                          EGF     43    17  35   9   45  20000                                          1X I/S/T                                                                             167    43  25  12   56  96000                                          __________________________________________________________________________

The effects of different lots of FCS, vitamin supplements (GIBCO MEMadditive), addition of 2-ME (50 μM) to I/S/T, and changing the growthmedium every other day (qod) instead of daily (qd) were observed and arereported in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    Effect of cell culture conditions                                             on thymic cell viability                                                             Cells                                                                         Per    %                                                               Culture                                                                              Fragment                                                                             Live                                                                              %     %     %                                               Condition                                                                            (×10.sup.3)                                                                    Cells                                                                             CD4+8-                                                                              CD4-8+                                                                              CD4+8+                                          __________________________________________________________________________    FCS#1  166    26  27     9    37                                              FCS#2  130    33  35    14    43                                              FCS#3  194    22  43    11    41                                              FCS#1  108    24  44    10    37                                              +2-me                                                                         FCS#1  116    44  48    12    35                                              +vitamins                                                                     FCS#1  144    12  38     9    46                                              qod                                                                           __________________________________________________________________________

In Table 2, data represent mean values averaged from five fragmentscultured under identical conditions for 12 days. Cultures were assayedon day 12 for cell yields, percent viability, and CD4+/8+ distributionpatterns. Different lots of FCS had little or no effect on thymus cellviability, and the addition of 2-ME to I/S/T-supplemented medium offeredno apparent advantage to culture maintenance. Vitamin supplementsappeared to confer some benefits in overall cell viability at day 12.Cultures fed every other day with I/S/T-supplemented medium had fewerviable cells than cultures fed every day. Based upon these results, itis preferred that thymic lobules be maintained with I/S/T-supplementedmedium that is replaced daily.

Example 2 Methods of Obtaining HIV for Infection of Thymic Lobules

PHA-activated T-cell blasts are required for preparing the HIV virusstocks. PHA blasts were either freshly prepared or frozen. Frozen cellsmust be thawed and put into culture media containing interleukin-2(IL-2) at least 24 hr before HIV infection. Fresh cells are preferableto frozen cells.

PHA-activated human T-cell blasts were produced by obtaining buffy coatsof human cells from a local blood bank. Cells were removed from the bagunder sterile conditions and mixed with an equal volume ofphosphate-buffered saline (PBS) or Hank's balanced salt solution (HBSS)with heparin (2 ml of 1000 units/ml made up in 250 ml of PBS or HBSS).Approximately 35 ml of the diluted cell suspension wad aliquoted into 50ml conical centrifuge tubes. The cell suspension was underlayered withapproximately 14 ml of Ficoll-hypaque (HISTOPAQUE®, Sigma) andcentrifuged at 450 RCF (about 1400 rpm) for 30 min with the brake off.The interface cells were harvested, washed twice with PBS and counted.In some cases, the cells were resuspended in 50 ml, diluted 1:5 andcounted. For incubation, cells were resuspended at 2-3×10⁶ per ml inIscove's Modified Dulbecco's (IMDM) media containing 10% FCS, 2 mML-glutamine, penicillin to a final concentration of 100 U/ml,streptomycin to a final concentration of 100 μg/ml and 1 μg/ml ofphytohemagglutinin-P (PHA-P). Cells were incubated, maintaining a celldensity of 2-3×10⁶ for three days and either frozen in 10% dimethylsulfoxide (DMSO)/FCS and stored in liquid nitrogen, or used directly ininfection experiments.

When frozen PHA blasts were used, the PHA blasts were thawed andincubated overnight in medium containing 50 units/ml of IL-2 prior toinfection. PHA blasts were then pelleted by centrifugation at 1000 rpmfor 5 min.

The PHA blasts were infected by resuspending the blasts in virus (10³-10⁴ TCID₅₀) containing 5 μg/ml of polybrene at 1-2×10⁷ cells/ml ofvirus. Viruses for infection must be thawed quickly at 37° C. and usedas soon as possible. The starting concentration of virus inoculum was 5ml of a stock with TCID₅₀ of 10³ or 10⁴ per 10⁸ cells. Infection wasallowed to occur for about 2 hr at 37° C. After 2 hr cells were dilutedup to the appropriate volume in medium containing IL-2 at 50 Units/ml ata cell density of 2-3×10⁶ /ml. On day 2 or 3 (depending on virus), thecells were pelleted and fresh medium was added. Starting on day 3 or 4,supernatant was collected until day 8 or 9 at 24-hr intervals, and freshmedium containing IL-2 was added. The collected supernatant wasaliquoted into freezing vials labelled with the date and type of virus.An aliquot also was saved for p24 analysis by adding 1/10 volume of 10%Triton X-100 to the supernatant and storing at 4° C. until analysis. Thefreezing vials containing virus were frozen immediately in liquidnitrogen.

Example 3 Infection Techniques

The "standard" method of infection of thymic organ cultures involvesdripping virus-containing PHA blast supernatant over the top of theorgan fragment, the so-called "drip" method. In the case of thymiclobules already deposited on Gelfoam® boats as described in Example 1,the effectiveness of this method was determined.

To increase the initial infectious dose at time zero, an alternativeinfection protocol, the "dip" method, was developed. In the dip method,thymic lobules were incubated for approximately 2 hours in vials ofundiluted virus-containing PHA blast supernatant. Dipped fragments weresubsequently transferred to Gelfoam® boats for incubation.

For infectivity analysis, tissue fragments were harvested and disruptedusing tissue homogenizers which allow for the release of lobule contents(thymocytes and other thymus constituents). Cells were counted andprocessed by: staining for flow cytometric analysis, quantitation of DNAcontent, immunohistochemistry, functional assays, and assessment ofviral replication.

The results of these experiments are summarized in FIGS. 3-7. Ingeneral, here and as described in the HIV-infected SCID-hu mouse,inoculation of TOC with HIV (Xho in this example) resulted in decreasedcell viability (FIG. 3), detectable p24 levels (FIG. 4), and decreasednumbers of CD4+CD8+ thymocytes (FIG. 5). Dip-infected TOC followedapproximately the same time course of HIV (Xho)-induced cell depletionas drip-infected cells and displayed lower viability by day 11post-infection, as shown in FIG. 3. Early in the infection, dip-infectedcells produce more p24 than drip-infected cells; by 11 dayspost-infection, however, p24 levels were the same for cells from lobulesinfected by either method (FIG. 4).

HIV p24 antigen was determined using a commercially available p24 ELISAkit (DuPont) according to the manufacturer's instructions. The resultsare presented as pg/10⁶ cells.

Dip-infected TOC showed a greater decrease in the CD4+8+ population(FIGS. 5(a) and (b)) and displayed a lower cellularity (FIG. 6) thandrip-infected lobules. The relative population of CD8+ cells was higherin dip-infected lobules than in drip-infected lobules, as shown in FIG.7. Thus, the dip method of infection appeared to increase the initialinfectious dose and to accelerate the spread of infection and the onsetof pathology compared to the drip method.

Example 4 Pathology of HIV in the System

Infection by HIV (SM isolate) established that the thymic lobule culturedisplayed typical indices of HIV pathology. TOCs were infected by thedrip method as described in Example 3. Cells from lobules were harvestedat varying times post-infection. FIG. 8 shows that p24 productionincreased significantly over the uninfected controls. Increased celldeath and decreased cellularity compared to uninfected controls is shownin FIGS. 9(a) and (b) respectively. Cell death and decreased cellularityare determined by decreased representation of cells with a high forwardscatter/low side scatter profile on FACS analysis. Time-dependentdepletion in the relative and absolute number of CD4+8+ thymocytes isshown in FIG. 10. The results indicate that the thymic lobule culturesystem displayed parameters associated with viral infection.

FACS analyses of infected cells from the lobules are shown in FIG. 11.HIV (Xho strain) infected thymic lobules (lower panel) had a lower cellviability (left panel) (as determined by decreased representation ofcells with a high forward scatter/low side scatter profile, typical oflive cells) and CD4+8+ population than uninfected controls (upperpanels).

Example 5 Comparison of Infectivity Parameters of Different Strains ofHIV

To determine if infection of the thymic organ system is reflective of invivo infectivity, Xho, NL4-3, EW, and RIP7 HIV isolates were tested. Theisolates were obtained as described in Example 2. Infection was carriedout by the dip method described in Example 3. The results are shown inTable 3. Data are mean values averaged from 6 fragments cultured underidentical conditions for 8 days.

The pathogenic effects of the Xho, NL4-3, and EW HIV isolates weresimilar, indicating that the thymic lobule culture system can be usedfor a variety of viral strains, thus expanding its applicability. Incontrast, the RIP7 strain, which grows well in PHA blasts in vitro anddoes not appear to replicate in SCID-hu Thy/Liv mice, did not grow wellin the thymic lobule culture system. These data, taken together, suggestthat the thymic lobule culture system more closely reflects the in vivoSCID-hu mouse system.

Example 6 Assay of Therapeutic Agents

To determine whether the cell culture system described in Example 1 waseffective as an in vitro model for screening potential anti-viralagents, known therapeutic agents were tested for their ability toinhibit viral infection. The virus used was HIV strain Xho. The knowndrugs were azidothymidine (AZT) and 2',3'-dideoxyinosine (ddI), both ofwhich suppress HIV infection in humans.

Varying amounts of AZT or ddI were added to lobules which had beeninfected with the HIV Xho strain by the dip method as described inExample 3. In this experiment, the growth medium contained no I/T/Ssupplement, as described in Example 1. Lobules were harvested at variousdays post-infection and their cellular contents were assayed for variousparameters of infection and pathology. One culture of control lobulesculture of control lobules was not infected and received no therapeuticagent. Parameters assayed were as follows: a) cell viability; b) p24content; and c) CD4+/8+ profiles. The results are shown in Table 4 andFIGS. 12-14.

The data indicate that both ddI and AZT inhibit viral replication in thethymic lobule culture system, even when administered at sub-micromolarlevels (Table 4). AZT treatment was inhibitory even at extremely lowdoses (0.001-0.01 μM), yet doses as high as 20 μM failed to completelyblock viral replication (Table 4 and FIGS. 12-14). In contrast, ddIdisplayed a nearly linear inhibition of viral replication as drug dosesincreased above the 2 μM range, and complete blockage was seen at 20 μM(Table 4 and FIGS. 12-14).

A comparison between the percent reduction in p24 values seen at variousdoses of AZT and ddI for three separate experiments is shown in Table 5.

The results presented in Table 5 suggest that the thymic lobule culturesystem can generate reproducible results reflecting the efficacy of bothAZT and ddI in blocking HIV replication.

                                      TABLE 3                                     __________________________________________________________________________                                 CD4                                              Days           p24 pg                                                                            Cells per to                   Abs. #                      post-    number of                                                                           per 10.sup.6                                                                      fragment                                                                           Percent                                                                            CD8                                                                              percent                                                                             percent                                                                             percent                                                                             4+8+                        infection                                                                          Virus                                                                             fragments                                                                           cells                                                                             ×10.sup.3                                                                    Live ratio                                                                            CD4+8-                                                                              CD4-8+                                                                              CD4+8+                                                                              ×10.sup.4             __________________________________________________________________________    8    none                                                                              6       0 610  70   1.60                                                                             7     5     86    53                          8    XHO 6     45707                                                                              93  37   0.69                                                                             13    19    53     5                          8    NL4-3                                                                             6     42229                                                                              83  25   0.60                                                                             12    21    48     4                          8    EW  6     11917                                                                             343  62   0.75                                                                             5     8     83    29                          8    RIP7                                                                              6      195                                                                              587  69   1.32                                                                             5     4     89    52                          __________________________________________________________________________

                                      TABLE 4                                     __________________________________________________________________________    Dose response of ddl and AZT (Xho Strain)                                                                      %                                            Days      Drug/                                                                              No. of                                                                             Cells Per    Reduction                Abs. #              Post- Virus                                                                             Dose Frag-                                                                              Fragment                                                                            Percet in p24                                                                              %      %     %     4+8+                Infection                                                                           (XHO)                                                                             (μM)                                                                            ments                                                                              ×10.sup.3                                                                     Live Cells                                                                           Value CD4+8- CD4-8+                                                                              CD4+8+                                                                              ×10.sup.4     __________________________________________________________________________    9     -   --   14   211   33     100   21     11    61    7                   9     +   --   14   111   14      0    24     25    32    2                   9     +   ddl  9    137   20      0    23     27    28    1                             0.002                                                               9     +   ddl  13   149   18     23    24     21    36    3                             0.020                                                               9     +   ddl  4    112   16     89    31     22    24    3                             0.200                                                               9     +   ddl 2.000                                                                          4    108   24     96    28     18    37    4                   9     +   ddl  4    170   21     100   19     11    63    11                            20.000                                                              9     +   AZT  5    107   16     22    16     27    37    2                             0.001                                                               9     +   AZT  9    110   15     66    27     22    22    2                             0.010                                                               9     +   AZT  4     99   23     82    26     21    24    2                             0.100                                                               9     +   AZT  4     91   20     86    32     22    24    2                             1.000                                                               9     +   AZT  9    112   22     91    25     18    46    4                             10.000                                                              9     +   AZT  5    167   17     90    18     17    58    6                             20.000                                                              __________________________________________________________________________

                                      TABLE 5                                     __________________________________________________________________________    Effect of ddI and AZT on Reduction of p24 Expression (Xho Strain)                    Percent Reduction in p24                                                      Values as Compared to                                                                      Percent Reduction in p24                                                                   Percent Reduction in p24                            Controls     Values as Compared to                                                                      Values as Compared to                        Drug/Dose                                                                            Experiment #1                                                                              Controls Experiment #2                                                                     Controls Experiment #3                       (μM)                                                                              (terminated at day 8)                                                                      (terminated at day 9)                                                                      (terminated at day 9)                        __________________________________________________________________________    none    0%           0%           0%                                          ddI 0.002                                                                            n.d.         n.d.          0%                                          ddI 0.020                                                                            n.d.         49%           0%                                          ddI 0.200                                                                            82%          89%          n.d.                                         ddI 20.000                                                                           99%          96%          n.d.                                         ddI 20.000                                                                           100%         100%         n.d.                                         AZT 0.001                                                                            n.d.         n.d.         22%                                          AZT 0.010                                                                            n.d.         73%          54%                                          AZT 0.100                                                                            89%          82%          n.d.                                         AZT 1.000                                                                            96%          86%          n.d.                                         AZT 10.000                                                                           93%          94%          87%                                          AZT 20.000                                                                           n.d.         n.d.         90%                                          __________________________________________________________________________

Two additional strains of HIV (EW and NL4-3) were also tested. Theinfection method, culture conditions, and controls were the same asdescribed for the Xho strain. Percent reduction in p24 levels forvarious doses of AZT and ddI is shown in FIGS. 15 (EW strain) and 16(NL4-3 strain). Several infection parameters from these dose responseexperiments are summarized in Tables 6 (EW strain) and 7 (NL4-3 strain).The results demonstrate that the thymic lobule culture system reflectsthe in vivo efficacy of both AZT and ddI in blocking replication of avariety of HIV strains.

It was also found that the effects of ddI and AZT more closelyapproximate those seen in vivo (i.e., with SCID-hu Thy/Liv mice orhumans) than in vitro (i.e., PHA-activated peripheral blood mononuclearcells (PBMC)). In contrast to the general observation in vitro, whereddI appears to be less effective than AZT in blocking HIV infection,both AZT and ddI appeared to have comparable efficacies in the thymiclobule culture system. These observations suggest that the thymic lobuleculture assay system more closely parallels the in vivo SCID-hu Thy/Livmodel than other in vitro cell assay systems for testing drug efficacyin blocking HIV infection.

                                      TABLE 6a                                    __________________________________________________________________________    Dose response of ddI and AZT (EW strain)                                                        Cells Per                                                                            p24         ratio   Abs #    %   %                           AZT                                                                              ddI                                                                              No. of                                                                            Fragment                                                                             pg/10.sup.6                                                                       p24 % HIV                                                                             CD  cells                                                                             4+8+ %   CD  CD                  Day Virus                                                                             μM                                                                            μM                                                                            lobes                                                                             ×10.sup.3                                                                      cells                                                                             + no drug                                                                             4 to 8                                                                            % live                                                                            ×10.sup.6                                                                    CD4+                                                                              4+8+                                                                              8+                  __________________________________________________________________________    9   NC  0  0  3   283      0 0       3.56                                                                              33  0.212                                                                              15.67                                                                             74.57                                                                             4.38                9   EW  0  0  3   207    21980                                                                             1       0.79                                                                              55  0.169                                                                              6.83                                                                              81.65                                                                             9.09                9   EW  0  0.01                                                                             3    73    34267                                                                             1.559   1.03                                                                              19  0.042                                                                              13.57                                                                             53.94                                                                             14.57               9   EW  0  0.1                                                                              3   186    4993                                                                              0.227   1.86                                                                              44  0.261                                                                              12.71                                                                             75.69                                                                             6.71                9   EW  0  1  3   413     409                                                                              0.019   2.97                                                                              44  0.343                                                                              10.52                                                                             82.78                                                                             3.56                9   EW  0  10 3   233      0 0       1.98                                                                              44  0.196                                                                              9.53                                                                              83.06                                                                             4.70                9   EW  0.01                                                                             0  3   140    22000                                                                             1.002   0.86                                                                              45  0.109                                                                              8.06                                                                              77.43                                                                             9.43                9   EW  0.1                                                                              0  3   280    2473                                                                              0.113   1.47                                                                              64  0.234                                                                              8.56                                                                              83.29                                                                             5.88                9   EW  1  0  3   387    1373                                                                              0.063   1.66                                                                              48  0.332                                                                              6.53                                                                              86.37                                                                             3.93                9   EW  10 0  3   173    8900                                                                              0.406   2.03                                                                              36  0.124                                                                              15.57                                                                             68.99                                                                             7.44                __________________________________________________________________________

                                      TABLE 6b                                    __________________________________________________________________________    Dose response of ddI and AZT (EW strain)                                                        Cells Per*                                                                           p24*        ratio*  Abs #*   %*  %*                          AZT                                                                              ddI                                                                              No. of                                                                            Fragment                                                                             pg/10.sup.6                                                                       p24 % HIV*                                                                            CD  cells*                                                                            4+8+ %*  CD  CD                  Day Virus                                                                             μM                                                                            μM                                                                            lobes                                                                             ×10.sup.3                                                                      cells                                                                             + no drug                                                                             4 to 8                                                                            % live                                                                            ×10.sup.6                                                                    CD4+                                                                              4+8+                                                                              8+                  __________________________________________________________________________    9   NC  0  0  3   188    0   0       0.57                                                                               9  0.14 3.33                                                                              4.20                                                                              0.41                9   EW  0  0  3   12     3451                                                                              0.157   0.17                                                                               6  0.02 1.74                                                                              5.54                                                                              3.61                9   EW  0  0.01                                                                             3   23     6768.6                                                                            0.3078  0.37                                                                              14  0.03 2.28                                                                              18.68                                                                             6.13                9   EW  0  0.1                                                                              3   188    2815.1                                                                            0.128   0.16                                                                               3  0.16 6.08                                                                              10.07                                                                             2.89                9   EW  0  1  3   201    272.71                                                                            0.023   0.42                                                                              19  0.17 1.13                                                                              2.70                                                                              0.25                9   EW  0  10 4   82     0   0       0.34                                                                              25  0.09 4.68                                                                              21.40                                                                             6.20                9   EW  0.01                                                                             0  3   34     2828.4                                                                            0.128   0.66                                                                              19  0.19 3.93                                                                              19.09                                                                             6.81                9   EW  0.1                                                                              0  3   106    1651.7                                                                            0.075   0.71                                                                              18  0.10 2.07                                                                              2.87                                                                              0.38                9   EW  1  0  3   23     721.76                                                                            0.033   0.57                                                                              13  0.02 0.65                                                                              1.41                                                                              1.05                9   EW  10 0  3   100    4902.9                                                                            0.223   0.27                                                                              19  0.10 2.31                                                                              8.95                                                                              4.13                __________________________________________________________________________     *These values represent standard deviations of corresponding rows in Tabl     6a.                                                                      

                                      TABLE 7a                                    __________________________________________________________________________    Dose response of ddI and AZT (NL4-3 strain)                                                     Cells Per                                                                            p24         ratio   Abs #    %   %                           AZT                                                                              ddI                                                                              No. of                                                                            Fragment                                                                             pg/10.sup.6                                                                       p24 % HIV                                                                             CD  cells                                                                             4+8+ %   CD  CD                  Day Virus                                                                             μM                                                                            μM                                                                            lobes                                                                             ×10.sup.3                                                                      cells                                                                             + no drug                                                                             4 to 8                                                                            % live                                                                            ×10.sup.6                                                                    CD4+                                                                              4+8+                                                                              8+                  __________________________________________________________________________    8   NL4-3                                                                             0  0  4   141      0 0       1.68                                                                              36  0.117                                                                              9.62                                                                              81.53                                                                             5.57                8   NL4-3                                                                             0  0   5*  36    24644                                                                             1       0.80                                                                              14  0.016                                                                              17.40                                                                             42.69                                                                             21.84               8   NL4-3                                                                             0  0.1                                                                              5   116    5001                                                                              0.203   2.01                                                                              36  0.081                                                                              16.25                                                                             69.93                                                                             8.31                8   NL4-3                                                                             0  1  5   142     907                                                                              0.037   1.50                                                                              49  0.119                                                                              7.79                                                                              83.96                                                                             5.21                8   NLA-3                                                                             0  10 5   102     61 0.002   2.41                                                                              42  0.079                                                                              13.60                                                                             77.94                                                                             5.53                8   NLA-3                                                                             0  100                                                                              5   195     17 0       3.13                                                                              42  0.153                                                                              13.92                                                                             78.33                                                                             4.57                8   NLA-3                                                                             0.1                                                                              0  5    71    1732                                                                              0.071   1.59                                                                              41  0.058                                                                              10.44                                                                             80.05                                                                             6.81                8   NL4-3                                                                             1  0  5   179    1007                                                                              0.041   1.85                                                                              52  0.153                                                                              8.60                                                                              84.57                                                                             4.56                8   NL4-3                                                                             10 0  5   142     978                                                                              0.041   2.36                                                                              35  0.111                                                                              13.36                                                                             77.32                                                                             5.72                8   NL4-3                                                                             100                                                                              0  5    83    1627                                                                              0.066   1.77                                                                              24  0.051                                                                              22.85                                                                             58.97                                                                             12.89                                 *Except p24 data is only 3 lobes.                           __________________________________________________________________________

                                      TABLE 7b                                    __________________________________________________________________________    Dose response of ddI and AZT (NL4-3 strain)                                                     Cells Per*                                                                           p24*        ratio*  Abs #*   %*  %*                          AZT                                                                              ddI                                                                              No. of                                                                            Fragment                                                                             pg/10.sup.6                                                                       p24 % HIV*                                                                            CD  cells*                                                                            4+8+ %*  CD  CD                  Day Virus                                                                             μM                                                                            μM                                                                            lobes                                                                             ×10.sup.3                                                                      cells                                                                             + no drug                                                                             4 to 8                                                                            % live                                                                            ×10.sup.6                                                                    CD4+                                                                              4+8+                                                                              8+                  __________________________________________________________________________    8   NC  0  0  4   55      0  0.00    0.49                                                                              6   0.05 4.34                                                                              5.82                                                                              0.89                8   NL4-3                                                                             0  0   5* 12     7377                                                                              0.30    0.08                                                                              3   0.01 3.67                                                                              13.85                                                                             4.17                8   NL4-3                                                                             0  0.1                                                                              5   63     415 0.02    0.52                                                                              6   0.04 2.53                                                                              2.57                                                                              1.44                8   NL4-3                                                                             0  1  5   18     343 0.01    0.05                                                                              8   0.02 0.45                                                                              1.51                                                                              0.40                8   NL4-3                                                                             0  10 5   18      35 0.00    1.05                                                                              7   0.02 6.50                                                                              7.94                                                                              0.42                8   NL4-3                                                                             0  100                                                                              5   102     15 0.00    0.88                                                                              3   0.08 1.86                                                                              0.97                                                                              0.72                8   NL4-3                                                                             0.1                                                                              0  5   39     369 0.02    0.33                                                                              15  0.03 0.39                                                                              3.03                                                                              1.87                8   NL4-3                                                                             1  0  5   47     181 0.01    0.42                                                                              7   0.05 3.03                                                                              3.81                                                                              0.86                8   NL4-3                                                                             10 0  5   94     333 0.01    0.32                                                                              2   0.07 0.54                                                                              0.44                                                                              0.63                8   NL4-3                                                                             100                                                                              0  5   62     1323                                                                              0.05    0.12                                                                              1   0.04 1.90                                                                              1.88                                                                              0.22                                  *Except p24 data is only 3 lobes.                           __________________________________________________________________________     *These values represent standard deviations of corresponding rows in Tabl     7a.                                                                      

We claim:
 1. A method of determining characteristics of viral infectionand pathology comprising:a. providing a culture of intact unstimulatedhuman thymic lobules; b. incubating the culture under conditionssuitable to maintain cell viability; c. exposing the lobules culture toa virus capable of infecting the lobules; d. observing the cells fromthe lobules for viral activity; and e. comparing the observations ofstep d with a hypothetical mechanism.
 2. The method according to claim 1wherein the thymic lobules are human fetal.
 3. The method according toclaim 1 wherein the thymic lobules are human neonate.
 4. The methodaccording to claim 1 wherein the thymic lobules are human adolescent oradult.
 5. The method according to claim 1 wherein the lobules arecontained in about 1 mm³ fragments containing 1-4 intact lobules.
 6. Themethod according to claim 1 wherein the initial culturing step is forabout 6-10 days.
 7. The method according to claim 1 wherein the lobulesare infected by incubation in a liquid containing the virus particlesfor a length of time suitable to allow infection.
 8. The methodaccording to claim 7 wherein the lobules are incubated in the liquid forapproximately 2 hours.
 9. The method according to claim 7 wherein thelobules are infected by dripping virus-containing liquid over thelobules.
 10. The method according to claim 1 wherein the virus used forinfection is selected from the group consisting of humancytomegalovirus, human immunodeficiency virus, herpes simplex virus, andrubella virus.